Various methods may be used in biology and in medicine to observe different targets in a biological sample. For example, analysis of proteins in histological sections and other cytological preparations may be performed using the techniques of histochemistry, immunohistochemistry (IHC), or immunofluorescence. Analysis of proteins in biological samples may also be performed using solid-state immunoassays, for example, using the techniques of western blots, or using cell-based assays that can be performed, for example, by using flow cytometry.
Many of the current techniques may detect only a few targets at one time (such as IHC or fluorescence-based Western blots where number of targets detectable is limited by the fluorescence-based detection system) in a single sample. Further analysis of targets may require use of additional biological samples from the source, limiting the ability to determine relative characteristics of the targets such as the presence, absence, concentration, and/or the spatial distribution of multiple biological targets in the biological sample. Moreover, in certain instances, a limited amount of sample may be available for analysis or the individual sample may require further analysis.
Methods of iteratively analyzing an individual sample are described in U.S. Pat. No. 7,629,125 and U.S. Pat. No. 7,741,046. In particular, U.S. Pat. No. 7,741,046 provides methods of detecting multiple targets in a biological sample that involve the use of oxidation for inactivating signal generators (e.g., for bleaching fluorescent dyes.) The oxidation reaction is accomplished by using oxidizing reagents, such as hydrogen peroxide. Additionally, a signal can be inactivated by continuous exposure of the signal generator to irradiation, i.e., by photobleaching. Similar to signal inactivation by oxidation, this process can be lengthy and may not proceed to completion, resulting in reduced signal-to-noise ratio. In addition, continued exposure of sample to irradiation may damage the biological sample.
However, often these methods of biomarker analysis are limited to detection of a relatively few markers (up to four) due to spectral overlap among available fluorophores and complex imaging systems requirements for hyperspectral imaging and deconvolution of spectra. For example, a multiplex immunofluorescence platform may utilize an antibody cycling process whereby a sample is stained with three antibodies per cycle, imaged, and restained with another set of antibodies after the signal from the first set of antibodies has been removed. This approach may be limited though, while the platform allows for the detection of multiple biomarkers, the repeated antibody incubations and destaining steps, which can range from 30 to 60 minutes per cycle, add a significant amount of time to the overall process.
US patent applications US20100120043A1 and US20110092381A1 describe a method of simultaneous hybridization of probes followed by sequential detection. After hybridization and detection of first set of probes the method includes steps of modifying the signal from the first set of probes followed by generating the signal from a second set of probes. To reduce process time, it would be of benefit to achieve the modification of one set of signal and activation of the next in the same step.
As such a method which provides a high throughput technology where repeated time-consuming probe incubation steps are diminished is desirable.